The long-range goal of this research program is the development of efficient in vivo gene delivery systems. The goal of this specific project is the development of an improved minimally invasive system for the delivery of plasmid DNA to the skin. Skin is easily accessibility which makes it an excellent target for gene therapy applications whether it is for directly treating cutaneous diseases or utilizing the skin as a depot for delivering proteins directly to the circulation for systemic therapy. To take advantage of the easy accessibility of the skin it is critical to develop non-contact approaches that are a simple and direct in vivo method to deliver DNA and can be accomplished in a minimally invasive way. We have been working on developing such approaches and previously developed devices and protocols that utilized electrotransfer for this delivery. While these devices have worked effectively and can accomplish this in a relatively non-invasive manner, it is still necessary to have contact between the electrodes and the tissue target. In addition, the applied voltages needed to achieve delivery on some occasions may cause cellular or tissue damage or potential discomfort. It is critical to develop an alternative approach that can work as well as electrotransfer but do it without the contact. An additional consideration is to develop an approach and/or device that will allow for better control of delivery and move towards a more predictable and reproducible pattern of expression. To accomplish this, we propose to utilize a non-thermal atmospheric pressure plasma device that can permeabilize cells and facilitate plasmid DNA uptake. We hypothesize that delivery is achieved by ion deposition on the surface of the target tissue and that if the level of ion deposition is regulated then expression levels can be controlled. The novel plasma device to be further developed and evaluated is based on nanosecond pulsed air plasmas. Using this approach will allow us to develop a small portable device that could be battery operated. This will be a non-contact delivery device that will minimize or eliminate potential discomfort and/or cellular damage. The following specific aims will be performed as part of this project. 1) To evaluate non-thermal atmospheric plasma devices for controlled production of ions that can be deposited on tissue surface; 2) To evaluate NTAP devices producing various level of ions for delivery of plasmid DNA to the skin and to determine the duration of maximal expression levels and to determine if this time can be increased by performing multiple delivery procedures; and 3) to determine if the system established in the first two aims can deliver plasmids encoding therapeutic proteins. The investigators have extensive expereince in developing non-thermal plasma devices and gene transfer so are well suited to successfully complete the study.